?-lactam antibiotics such as the penicillins and cephalosporins are the most often used antibiotics and account for more than 60% of total world consumption of antimicrobials. Due to widespread ?-lactam antimicrobial use, bacterial resistance has been increasing and now represents a serious threat to the continued use of antibiotic therapy. The current situation with hospital-associated infections resulting from antibiotic resistant gram-negative rods is critical in that no new drugs are expected in the near future to treat these infections. Resistance rates have been increasing for several gram-negative species and multidrug resistance is a particular problem in that some clinical strains are resistant to many classes of antibiotics; leaving few options for treatment. The most common mechanism of bacterial resistance to ?-lactam antibiotics is the synthesis of ?-lactamases that hydrolyze the drugs to generate ineffective products. ?-lactamases are classified into four groups A, B, C and D based on amino acid sequence homologies. Class A ?-lactamases are widespread in both gram-positive and gram-negative bacteria and exhibit broad substrate hydrolysis profiles which include penicillins, cephalosporins and, for a few enzymes, carbapenems. The class A TEM-1 and SHV-1 ?-lactamases are common plasmid-encoded ?-lactamases in gram-negative bacteria and are a widespread source of antibiotic resistance. The class A KPC b-lactamase has emerged in K. pneumoniae and other gram-negative rods in recent years and is a cause for concern due to its broad substrate profile that includes virtually all ?-lactam antibiotics including carbapenems. Adding to the concern is the difficulty in diagnosing infections with carbapenem resistance mediated by KPC. The ?-lactamase inhibitory protein (BLIP) is a 165 amino acid protein produced by Streptomyces clavuligerus which binds and inhibits several class A ?-lactamases. The goal of the project is to develop a BLIP-based protein reagent that can be used to specifically identify the KPC enzyme while not binding to other class A ?-lactamases such as the common TEM-1 and SHV-1 enzymes. In particular, the proposed experiments will utilize the class A ?-lactamase binding profile of BLIP in combination with a recently developed genetic screen to tailor the BLIP recognition properties to create variants that can uniquely recognize KPC ?-lactamase and thereby gain detailed information on the antibiotic resistance potential of clinical isolates that can be used to guide treatment and infection control strategies. In addition, the work will guide future studies using the proposed approaches for the development of similar assays targeting other emerging ?-lactamases. PUBLIC HEALTH RELEVANCE: This project addresses the need for identification of KPC ?-lactamase-mediated antibiotic resistance in gram-negative bacteria. ?-lactamases catalyze the destruction of b-lactam antibiotics and are the most common mechanism of resistance to these drugs. The proposed experiments will create an engineered version of the ?-lactamase inhibitory protein that is able to specifically recognize the clinically important KPC ?-lactamase and thereby can serve as an efficient diagnostic reagent to guide treatment and infection control strategies.